Box 1: Cyclotide synthesis

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Although synthetic macrocycle chemistry has been inspired
by the study of natural products such as cyclosporine,
the biosynthetic pathways for many compounds remain a mystery. Now, a team led
by Professor James Tam at Nanyang
Technological University has identified a plant-produced
peptide ligase that could be developed as a tool to synthesize cyclic peptides.3

Tam has long studied plants used in traditional Chinese medicine to
identify bioactive compounds, and he has previously characterized members from
a large and diverse family of plant-produced cyclic peptides known as
cyclotides. His team sought to identify the enzyme responsible for cyclotide production in a medicinal plant, Clitoria
ternatea.

To identify the peptide ligase that could be responsible for producing
the cyclotides in C. ternatea, the team used liquid chromatography and
fluorescent reporter constructs to isolate the activity and identify and
sequence the protein responsible, which they named butelase 1.

The enzyme
was purified and shown to efficiently ligate or cyclize almost any N-terminal
residue to a C-terminal asparagine as long as the asparagine is encoded in an
asparagine-histidine-valine sequence. The ligase also worked on non-native
substrates with the tag appended.

Boston University associate
professor Adrian Whitty told SciBX, "The impressive thing about this
work is that they were able to find an enzyme that was so broad in its
reactivity it requires an asparagine in one of the litigation positions but
accepts almost any amino acid in the other, which allows for great freedom of
design."

PeptiDream Inc.
CSO Patrick Reid said that adapting this technology for peptide-library
generation is not trivial, so it is not yet clear whether the ligase will have
utility for large-scale compound synthesis.

A first
step will be demonstrating that a recombinant form of the enzyme can be
expressed and purified.

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